Dental Pulp Stem Cells: An Attractive Alternative for Cell Therapy in Ischemic Stroke

You mean your mentors and senior researchers are so fucking incompetent they missed this from May 2016? My god, is there a need for a smidgen of stroke leadership.

TOOTH (The Open study Of dental pulp stem cell Therapy in Humans): Study protocol for evaluating safety and feasibility of autologous human adult dental pulp stem cell therapy in patients with chronic disability after stroke May 2016

 

Dental Pulp Stem Cells: An Attractive Alternative for Cell Therapy in Ischemic Stroke

Xiaoyan Lan¹, Zhengwu Sun², Chengyan Chu¹, Johannes Boltze^3† and Shen Li^1*†

• ¹Department of Neurology, Dalian Municipal Central Hospital Affiliated to Dalian Medical University, Dalian, China
• ²Department of Pharmacy, Dalian Municipal Central Hospital Affiliated to Dalian Medical University, Dalian, China
• ³School of Life Sciences, University of Warwick, Coventry, United Kingdom

Ischemic stroke is a major cause of disability and mortality worldwide, but effective restorative treatments are very limited at present. Regenerative medicine research revealed that stem cells are promising therapeutic options. Dental pulp stem cells (DPSCs) are autologously applicable cells that origin from the neural crest and exhibit neuro-ectodermal features next to multilineage differentiation potentials. DPSCs are of increasing interest since they are relatively easy to obtain, exhibit a strong proliferation ability, and can be cryopreserved for a long time without losing their multi-directional differentiation capacity. Besides, use of DPSCs can avoid fundamental problems such as immune rejection, ethical controversy, and teratogenicity. Therefore, DPSCs provide a tempting prospect for stroke treatment.

The past decade has witnessed intense advancement and tremendous therapeutic achievements in the ability to diagnose and treat stroke, a cerebrovascular disease of which 87% is ischemic in nature. Nevertheless, stroke remains a major cause of disability, morbidity, and mortality worldwide, and constitutes a major socioeconomic problem (1, 2). Ischemic stroke, due to partially or completely blocked blood flow in a cerebral artery, causes ischemic necrosis of brain tissue seriously impairing the health of affected individuals. The main therapeutic strategy for ischemic stroke is timely recanalization. This can either be achieved by tissue-type plasminogen activator application or mechanical thrombectomy. Particularly the latter can be applied up to 24 h after stroke in patients exhibiting a penumbra, and has revolutionized acute stroke treatment. However, the absolute number of patients qualifying for recanalization remains very low (3, 4). Hence, additional treatment approaches being effective beyond the first hours after stroke onset are urgently required.

Stem cell transplantation is a promising strategy to restore neurological function after stroke (5). Experimental stem cell transplantation in animals showed that numerous cell populations can improve functional recovery by a broad spectrum of mechanisms (6–8). Several kinds of stem cells are currently considered for therapy. These include embryonic stem cells (ESCs), fetal stem/progenitor cells, induced pluripotent stem cells (iPSCs), and adult stem cells. While embryonic or induced pluripotent stem cells exhibit a tremendous differentiation potential, they may also inherit a risk for tumor formation (9). The use of embryonic stem cells or fetal stem/progenitor cells raises ethical concerns. Adult stem cells show a limited proliferation and differentiation potential, but can still be beneficial after stroke due numerous mechanisms beyond tissue restoration. They are further believed to be safer in clinical application and their use is ethically less challenging (9–13).

Recent systematic reviews and meta-analyses on the most prominent adult stem cell therapy candidates, mesenchymal stem cells (MSCs), presented evidence that MSCs improve the outcome after stroke in animals (14) and patients, and confirmed the safety and feasibility of the approach (15). Nevertheless, there is still a lack of adult (stem) cells that can be derived from an autologous source, and may exhibit therapeutic abilities beyond those of MSCs.

Dental Pulp Stem Cells (DPSCs): A New Source of Adult Stem Cells

The dental pulp is a soft tissue located in the center of teeth. It comprises blood vessels, neural fibers, and connective tissue. The dental pulp contains both mesenchymal and ectodermal tissue as well as neural crest cells (16). Limited dentinal repair in the postnatal organism relies on specialized precursor cell populations residing in the dental pulp tissue. Gronthos et al. first reported the isolation and characterization of stem cells from dental pulp tissue of the third molar in 2000 (17). DPSCs are ectoderm-derived stem cells, originating from migrating neural crest cells (Figure 1). They are a subpopulation among dental pulp cells (DPCs) which possess MSC properties, such as a fibroblast-like morphology, adherence to a plastic surface, as well as surface marker expression, proliferation and colony forming behavior similar to that of MSCs (18, 19). It is not clear whether or not DPSCs are a kind of MSC population. Given their differentiation abilities as reviewed below, it might be assumed that DPSCs are a more naïve stem cell population that also, but not exclusively, exhibits MSC properties. A major benefit of DPSCs is that they can be isolated during routine dental procedures such as the eruption of deciduous teeth or extraction of impacted wisdom teeth (20) in simple and autologous fashion without ethical concerns. Another primary advantage of DPSCs is their potential for cell banking. Several studies have demonstrated that DPSCs retain their stem cell properties after long cryopreservation (21, 22). This is essential as cryopreservation can impact therapeutic capacities of other adult stem cell-containing populations in stroke (23). In addition, DPSC cultures can be established from extracted human molars with high efficiency, even after the whole tooth has been cryopreserved for up to 1 month (24). DPSCs also exhibit a multilineage differentiation potential into chondrocytes, adipocytes, odontoblasts, and potentially even neural-like cells (25–28).

FIGURE 1

Figure 1. DPSCs origin, isolation, and marker expression. DPSCs originate from migrating neural crest cells, coming to rest in dental pulp, and express markers overlapping with MSCs, ESCs, and NCSs.

Currently, there are no specific markers that uniquely define DPSCs. In general, DPSCs, as a heterogeneous population, express a variety of markers similar to MSCs (Table 1) (Figure 1), and do not express hematopoietic markers such as CD14, CD19, CD34, and CD45 (18, 26, 29–32). DPSCs isolated by their high proliferative potential tend to include a large population of cells expressing CD44⁺, CD90⁺, and CD166⁺. However, DPSCs also express stemness-related markers similar to ESCs such as Oct-3/4, Nanog, and Sox-2, as well as the cytoskeleton-related markers nestin and vimentin (Figure 1) (33–35). They further express insulin-like growth factor 1 receptor (IGF1R) which is regarded as a pluripotency marker in ESCs. DPSC-secreted IGF1 interacts with IGF1R through an autocrine signaling pathway to maintain self-renewal and proliferation potential (36).

TABLE 1

Table 1. Characteristics of DPSCs.

In addition, DPSCs (as neural crest-derived stem cells) not only express a number of neural stem cell (NSC) associated markers including nestin (26, 37) and Sox2 (38) (Figure 1), but also express low basal levels of markers associated with mature central nervous system cell types, including the neuronal markers βIII-tubulin, microtubule-associated protein 2 (MAP2), neurofilaments (NF) (33, 39), NeuN (40), the astrocytic marker glial fibrillary acidic protein (GFAP) (26, 33), and oligodendrocyte-associated CNPase (33). Taken together, this suggests that DPSCs can indeed differentiate into neuron-like cells under appropriate conditions, and differentiated cells even exhibit typical electrophysiological properties after neuronal differentiation (41, 42).

DPSCs as a Potential Candidate for Therapy of Neurological Diseases

Brain-derived NSCs are considered a promising population for stroke treatment due to their ability to self-renew and to differentiate into neural cells types (neurons, astrocytes, oligodendrocytes) (43). However, autologous harvest of adult human NSCs requires neurosurgical procedures due to their brain parenchymal residence (44), while allogeneic or even xenogenic NSCs grafting imposes the risk of graft rejection and additional immunological damage. Only a limited number of clinical trials currently explore the potential of NSCs for stroke treatment because of these limitations.

Adult stem cells or stem cell-containing populations are more frequently applied in translational research. As stated above, DPSCs share many biological characteristics with MSCs including bone marrow MSCs (BM-MSCs), adipose tissue-derived stem cells (ADSCs) and umbilical cord MSCs (UC-MSCs) but there are some variations in their proliferation potential (17, 27, 45), differentiation potential (17, 27, 46), immunomodulatory activity (27), secretome characteristics, and secretory capacity (47–49). Specifically, DPSCs have a higher proliferation rate and a greater clonogenic potential than MSCs (17, 45). Next to DPSCs, the DPC population also contains a higher number of stem/progenitor cells as compared to bone marrow (50). This may be attributed to the developmental state of the respective tissues. All teeth, even the permanent molars, are generated early in individual development and rest in the jar until they erupt. Abilities and capacities of stem cells may be much better preserved in tissue with a slow turnover such as the dental pulp when compared to BM, which exhibits a tremendous turn-over throughout life apart from some niches. DPSCs maintain their high rate of proliferation even after extensive subculturing.

Like MSCs, DPSCs can differentiate into cells of mesenchymal and non-mesenchymal tissues in vitro and in vivo. However, DPSCs exhibit stronger odontogenesis and neurogenesis capabilities, in turn being not as potent to produce adipogeneic, osteogeneic and chondrogeneic tissue than BM-MSCs (51) and ADSCs (46). Besides, DPSCs also have immunomodulatory capacities exceeding those of BM-MSCs, for example a higher suppression rate of T lymphocyte growth (17, 27).

DPSCs exhibit superior neuroprotective and neuro-supportive properties in neurological injuries and pathologies as compared with BM-MSCs and ADSCs (52). This might be related to a higher expression of trophic factors including brain derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF) in DPSCs as compared to BM-MSCs (47, 48), although the spectrum of growth and trophic factors secretion is similar (53). DPSCs also express higher quantities of CXCL14 and monocyte chemoattractant protein 1 (MCP-1) than ADSCs (49). Besides, the DPSC secretome contains higher concentrations of RANTES, FRACTALKINE, fms-related tyrosine kinase 3 (FLT-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and MCP-1 than the BM-MSCs secretome (54). DPSCs show higher angiogenic and neurogenic potentials in ectopic transplantation models compared to BM-MSCs and ADSCs, and exhibit the highest migration capacity. Transplantation of DPSCs in a mouse hindlimb ischemia model produced higher blood flow and capillary density than transplantation of BM-MSCs and ADSCs, which being associated with superior recovery of limb movement abilities and reduction of ischemic hindlimb damage (55). DPSCs also mediate stronger anti-apoptotic effects in a microenvironment challenged by oxidative and serum deprivation than BM-MSCs, ADSCs and UC-MSCs (45).

Cell size and diameter are important for safety after intravascular delivery as they are the major, but not the only, determinants of vascular obstruction and complications (56) (Table 2). Previously reported studies showed that the cell diameter of human DPSCs is around 15–16 μm (59), which is comparable to NSCs (57, 58) but slightly smaller than for most MSC populations (57, 60) including human BM-MSCs (61). Still this means that one has to expect a considerable pulmonary passage filtering effect after intravenous delivery, as well as a risk for microembolism after intraarterial administration (62). Hence, thorough investigations identifying the optimal route of DPSC administration by considering safety and efficacy aspects are recommended in DPSC translational research.